Alterations of circulating bone marrow-derived VEGFR-2+ progenitor cells in isolated limb perfusion with or without rhTNF-α.

BACKGROUND: Circulating endothelial progenitor cells (cEPCs) as recruited to the angiogenic vascular system of malignant tumors have been proposed as a biomarker in malignancies. The effect of antitumor chemotherapy on cEPCs is not fully understood. We examined the level of cEPCs, vascular endothelial growth factor (VEGF), and angiopoietin-2 in the blood of sarcoma and melanoma patients before and after isolated limb perfusion (ILP) with or without recombinant human tumor necrosis factor-α (rhTNF-α). METHODS: Twenty-two patients, 11 each with soft tissue sarcoma or recurrent melanoma of the limb, were recruited. ILP was performed with rhTNF-α/melphalan (TNF) or melphalan only (no TNF). Fifteen healthy volunteers served as control subjects. Blood was sampled before and up to 6 weeks after ILP. Peripheral blood mononuclear cells were isolated by density gradient centrifugation, and annexin V-negative cells were characterized as cEPCs by triple staining for CD133(+), CD34, and VEGFR-2(+). RESULTS: Before treatment, cEPC numbers were significantly increased in sarcoma (0.179 ± 0.190 %) and melanoma patients (0.110 ± 0.073 %) versus healthy controls (0.025 ± 0.018 %; P < 0.01), but did not differ significantly between sarcoma and melanoma patients. cEPC decreased significantly after ILP in patients with no TNF compared to pretreatment values (P < 0.05) and were significantly lower at 4 h, 48 h, and 1 week compared to ILP with TNF (P < 0.05). Values 6 weeks after ILP were significantly lower than before ILP in both investigated groups (P < 0.01). CONCLUSIONS: ILP with TNF results in activation of bone marrow-derived EPCs compared to ILP without TNF. Alteration of cEPCs and angiopoietin-2 by rhTNF-α might account for the cytotoxicity and hemorrhagic effects on tumor vessels during limb perfusion procedures.

Increased levels of circulating endothelial progenitor cells in patients with Moyamoya disease.

BACKGROUND AND PURPOSE: Chronic cerebral ischemia leads to higher risk for strokes attributable to insufficient collateralization, resulting from inadequate capacity for arteriogenesis and angiogenesis. Patients with Moyamoya disease (MMD) have similar transient ischemic attack frequencies compared to patients with chronic cerebral ischemia with other etiologies, but a strong capacity for arteriogenesis and angiogenesis. The mechanisms involved in the upregulation of the arteriogenesis and angiogenesis in MMD still remain unknown. In the present study we investigated if circulating endothelial progenitor cells are increasingly mobilized during MMD. METHODS: Twenty MMD patients, 8 patients with atherosclerotic cerebrovascular disease, and 15 healthy individuals were included in this study. Peripheral blood mononuclear cells were isolated by Ficoll density gradient centrifugation and circulating endothelial progenitor cells were characterized by triple staining using antibodies against CD133, CD34, and vascular endothelial growth factor receptor-2. Serum concentrations of vascular endothelial growth factor and granulocyte-macrophage colony-stimulating factor were determined by enzyme-linked immunosorbent assay. RESULTS: In MMD patients the number of circulating endothelial progenitor cells was significantly higher than in atherosclerotic cerebrovascular disease patients (P<0.002) and healthy controls (P<0.0001). Serum vascular endothelial growth factor concentrations in MMD patients and in atherosclerotic cerebrovascular disease patients were significantly higher compared to those in healthy controls (P<0.0001). Similar findings were observed for granulocyte-macrophage colony-stimulating factor. An inverse correlation between circulating endothelial progenitor cell numbers and serum levels of vascular endothelial growth factor (r=-0.53; P<0,02) was found in the MMD group. CONCLUSIONS: Our results show increased circulating endothelial progenitor cell numbers in MMD, which may play a role in the increased arteriogenesis and angiogenesis in MMD. Moreover, our results suggest that increased circulating endothelial progenitor cell mobilization in MMD may not be entirely mediated by vascular endothelial growth factor or granulocyte-macrophage colony-stimulating factor.

CXCR-4 expression by circulating endothelial progenitor cells and SDF-1 serum levels are elevated in septic patients.

BACKGROUND: Endothelial progenitor cell (EPC) numbers are increased in septic patients and correlate with survival. In this study, we investigated, whether surface expression of chemokine receptors and other receptors important for EPC homing is upregulated by EPC from septic patients and if this is associated with clinical outcome. METHODS: Peripheral blood mononuclear cells from septic patients (n = 30), ICU control patients (n = 11) and healthy volunteers (n = 15) were isolated by Ficoll density gradient centrifugation. FACS-analysis was used to measure the expression of the CXC motif chemokine receptors (CXCR)-2 and - 4, the receptor for advanced glycation endproducts (RAGE) and the stem cell factor receptor c-Kit. Disease severity was assessed via the Simplified Acute Physiology Score (SAPS) II. The serum concentrations of vascular endothelial growth factor (VEGF), stromal cell-derived factor (SDF)-1α and angiopoietin (Ang)-2 were determined with Enzyme linked Immunosorbent Assays. RESULTS: EPC from septic patients expressed significantly more CXCR-4, c-Kit and RAGE compared to controls and were associated with survival-probability. Significantly higher serum concentrations of VEGF, SDF-1α and Ang-2 were found in septic patients. SDF-1α showed a significant association with survival. CONCLUSIONS: Our data suggest that SDF-1α and CXCR-4 signaling could play a crucial role in EPC homing in the course of sepsis.

Clinical review: immunomodulatory effects of dopamine in general inflammation.

Large quantitaties of inflammatory mediators are released during the course of endotoxaemia. These mediators in turn can stimulate the sympathetic nervous system (SNS) to release catecholamines, which ultimately regulate inflammation-associated impairment in tissue perfusion, myocardial impairment and vasodilatation. Treatment of sepsis is based on surgical and/or antibiotic therapy, appropriate fluid management and application of vasoactive catecholamines. With respect to the latter, discussions on the vasopressor of choice are still ongoing. Over the past decade dopamine has been considered the 'first line' vasopressor and is frequently used to improve organ perfusion and blood pressure. However, there is a growing body of evidence that dopamine has deleterious side effects; therefore, its clinical relevance seems to be more and more questionable. Nevertheless, it has not been convincingly demonstrated that other catecholamines are superior to dopamine in this respect. Apart from its haemodynamic action, dopamine can modulate immune responses by influencing the cytokine network. This leads to inhibition of expression of adhesion molecules, inhibition of cytokine and chemokine production, inhibition of neutrophil chemotaxis and disturbed T-cell proliferation. In the present review we summarize our knowledge of the immunomodulatory effects of dopamine, with an emphasis on the mechanisms by which these effects are mediated.

Circulating endothelial progenitor cells in malignant gliomas.

OBJECT: Recent experimental work suggests that circulating endothelial progenitor cells (cEPCs) are recruited to the angiogenic vascular system of malignant gliomas. Consequently, the level of cEPCs has been proposed as a novel biomarker for the diagnosis and monitoring of these lesions. The aim of the present study was to examine the level of cEPCs and the level of EPC mobilizing mediators in the blood of patients with malignant gliomas. The authors were also interested in whether a correlation could be observed between the level of cEPCs and the extent of glioma angiogenesis determined by conventional methods. METHODS: Peripheral blood mononuclear cells from the whole blood of 12 patients with malignant gliomas (all glioblastomas multiforme [GBMs]), 10 with metastases to the brain, and 10 healthy volunteers were isolated using Ficoll density gradient centrifugation. The number of cEPCs was quantified by fluorescence-activated cell sorting analysis using antibodies against CD34, CD133, and VEGFR-2. Serum concentrations of VEGF and granulocyte-macrophage colony-stimulating factor (GM-CSF) were determined using the enzyme-linked immunosorbent assay. Histological analysis of tumor blood vessel density was performed by CD34 immunohistochemical staining. RESULTS: The number of cEPCs was significantly higher in patients with GBMs than in those with metastases (p < 0.04) or in the healthy volunteers (p < 0.004). The serum VEGF concentrations in patients with GBMs and metastases were significantly higher than in the healthy volunteers (p < 0.0001). Similar findings were observed for concentrations of GM-CSF. In addition, the patients in the GBM group with higher levels of cEPCs had significantly higher tumor blood vessel densities (1.71 +/- 1.17% of total area) compared with patients who had low levels of cEPCs (0.62 +/- 0.28% of total area; p < 0.02). CONCLUSIONS: Endothelial progenitor cells are increasingly mobilized in patients with malignant gliomas, and their levels correlate with tumor angiogenic activity. Therefore, the authors' results suggest that cEPCs may have the potential to serve as a novel biomarker for the identification of patients who would benefit from antiangiogenic therapy for GBM.

Circulating endothelial progenitor cells are increased in human lung cancer and correlate with stage of disease.

AIM: Recent studies suggest that circulating endothelial progenitor cells (cEPCs) are recruited to the angiogenic vascular system of non-small-cell lung cancer (NSCLC) and correlate with clinical behaviour. Consequently, the level of cEPCs has been proposed as a novel biomarker for disease progression in NSCLC. The role of cEPCs for the vascularisation of small-cell lung cancer (SCLC) is still unknown. Therefore, this study aims to examine the level of cEPCs as well as the level of EPC mobilising mediators in the blood of lung cancer patients and the correlation with tumour stage and disease progression. METHODS: In this study, 36 patients with biopsy-proven lung cancer (32 NSCLC, 4 SCLC) and 15 healthy individuals were recruited. Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll density gradient centrifugation, and cEPCs were characterised by triple staining using antibodies against CD133, CD34 and vascular endothelial growth factor-receptor (VEGFR)-2. Serum concentrations of VEGF were determined by enzyme-linked immunosorbent assay (ELISA). RESULTS: In lung cancer patients, the number of cEPCs was significantly higher than in healthy controls (p=0.000). Regarding tumour stage, NSCLC patients with UICC III-IV had significantly higher EPC counts than UICC I-IIB patients (p=0.044). Serum VEGF concentrations in lung cancer patients were significantly higher than in healthy controls (p=0.000) and correlated with cEPC numbers for all the groups (r=0.42, p=0.003). Follow-up data (n=20) revealed significantly higher cEPC numbers in lung cancer patients with tumour progression than in patients without evidence or progression of disease. The relative change in cEPC numbers between pre- and post-treatment assessment was significantly correlated to disease progression (p=0.000, log rank test) and the combined end points of progression and/or death (p=0.003, log rank test). CONCLUSION: Our results show increased cEPCs numbers in lung cancer patients, which may play a role in the vascularisation of lung cancer. Moreover, our results suggest an association of cEPC numbers with tumour stage and progression.

Increased circulating endothelial progenitor cells in septic patients: correlation with survival.

OBJECTIVE: Endothelial damage and detachment of endothelial cells are known to occur in septic patients. Thus, recruitment of circulating endothelial progenitor cells (cEPCs) to these lesions might have a beneficial effect on the clinical course in septic patients. Therefore, we were interested in whether EPCs, detected by flow cytometry, are increasingly mobilized during sepsis and if this mobilization is associated with clinical outcome. DESIGN: Prospective, nonrandomized study. SETTING: Intensive care unit of a university hospital. PATIENTS: Patients with (n = 32) and without (n = 15) sepsis and healthy volunteers (n = 15). INTERVENTIONS: Peripheral blood mononuclear cells were isolated by Ficoll density gradient centrifugation, and cEPCs were characterized by three-color fluorescence flow cytometry using antibodies against CD133, CD34, and vascular endothelial growth factor receptor-2. Serum concentrations of vascular endothelial growth factor, granulocyte macrophage-colony stimulating factor, and erythropoietin were determined by enzyme-linked immunosorbent assay. Severity of sepsis was assessed according to Acute Physiology and Chronic Health Evaluation II scoring. MEASUREMENTS AND MAIN RESULTS: In septic patients, the number of cEPCs was significantly higher than in nonseptic intensive care unit patients (p < .05) and healthy controls (p < .02). Nonsurvivors (n = 8), defined as death within 28 days after onset of sepsis, had significantly lower numbers of cEPCs than survivors (n = 24) (p < .0001). The number of cEPCs was correlated with survival in septic patients. Serum vascular endothelial growth factor concentrations were significantly higher in septic patients compared with nonseptic intensive care unit patients and healthy controls (p < .01) and correlated with the cEPC numbers (p < .0001). Similar findings were observed for granulocyte macrophage-colony stimulating factor and erythropoietin. CONCLUSIONS: Our data suggest that cEPC enumeration in peripheral blood of septic patients might be a valuable marker to assess the clinical outcome in these patients.

Secreted phospholipases A2 induce the expression of chemokines in microvascular endothelium.

Acute respiratory distress syndrome (ARDS) is characterized by alterations in microvascular permeability. In ARDS secreted phospholipase A(2) (sPLA(2)) IB and IIA are found to be highly upregulated. In this study, we therefore investigated the influence of exogenously added sPLA(2)-IB and sPLA(2)-IIA on the production of chemokines and adhesion molecules in lung microvascular endothelial cells (LMVEC). Treatment of LMVEC with sPLA(2)s resulted in a significant increase in the production of chemokines and adhesion molecules due to an increased expression of their mRNA and in an enhanced release of oleic acid. The upregulation of chemokines and adhesion molecules by LPS was stronger in the presence of sPLA(2). Activation of NF-kappaB occurred upon stimulation with sPLA(2). Moreover the MAPkinase pERK seems to be involved since a specific pERK inhibitor, e.g., U0126, but not a p38Kinase inhibitor, e.g., SB203580 prevented sPLA(2)-induced chemokine upregulation. Our data therefore suggest that LMVEC are a highly sensitive target for the direct action of extracellular sPLA(2)s.

Amelioration of endotoxin-induced sepsis in rats by membrane anchored lipid conjugates.

OBJECTIVE: In the pathogenesis of septic shock, caused by either bacterial toxins or trauma, increased production of multiple proinflammatory mediators, such as phospholipase A(2) (PLA(2)), cytokines, and chemokines, is known to be of major importance. The present study was undertaken to investigate the influence of a newly designed extracellular PLA(2) inhibitor (ExPLI) on synthesis of proinflammatory mediators and mortality rate in a rat sepsis model. DESIGN: Prospective, randomized animal study. SETTING: Experimental laboratory. SUBJECTS: Male Wistar-rats weighing 200-300 g. INTERVENTIONS: Mortality was induced by intraperitoneal bolus administration of lipopolysaccharide 15 mg/kg in 22 rats that were pretreated with NaCl or ExPLI (150 mg/kg). Furthermore, nine rats received a sublethal bolus of lipopolysaccharide (7.5 mg/kg) and nine rats received lipotechoic acid (8 mg/kg) simultaneously with or after ExPLI administration. Blood samples were collected from these rats, and cytokine concentrations were assessed by enzyme-linked immunosorbent assay. Lung and kidney were removed for RNA isolation and immunohistological analysis. MEASUREMENTS AND MAIN RESULTS: ExPLI treatment significantly reduced lipopolysaccharide-induced mortality of rats (90.9 and 36.4%, p <.05). Up-regulation of tumor necrosis factor-alpha and interleukin-6 production in serum after endotoxin treatment was significantly inhibited when ExPLIs were administered at the time of or before sepsis induction by using lipopolysaccharide or lipotechoic acid (p <.01). Similarly, messenger RNA expression of secreted PLA(2)-IIA, interleukin-1, or inducible nitric oxide synthase and the expression of intercellular adhesion molecule-1 were significantly down-regulated in lung and kidney of ExPLI-treated rats, as demonstrated by RNase protection assay, reverse transcription-polymerase chain reaction, or immunohistochemistry. CONCLUSIONS: ExPLIs may be considered as potentially effective compounds to prevent the production of inflammatory mediators and to improve mortality rate in septic patients.